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Updated: Jun 21, 2026
In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells
Published on: May 16, 2011
Virulence Is More than Adhesion and Invasion Ability, an In Vitro Cell Infection Assay of Bovine Mycoplasma spp
Elhem Yacoub1, Daniel Kos2, Murray Jelinski1
1Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5A2, Canada.
Abstract:
Mycoplasma bovis is the most common mycoplasma associated with cattle diseases worldwide. However, other seemingly less virulent Mycoplasma spp. such as M. bovigenitalium and M. bovirhinis have also been associated with mycoplasmosis. The study objective was to compare the adhesion and cellular invasion characteristics of these bovine Mycoplasma spp. using Madin-Darby Bovine Kidney (MDBK) epithelial cells. MDBK cells were separately infected with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis. Following infection, a gentamicin protection assay was performed and the cells lysed at 6 and 54 h post-infection. The MDBK cell lysates were cultured for Mycoplasma spp. and qPCR was used to estimate the average number of Mycoplasma bacterial cells that infected each MDBK cell (Myc/Cell ratio). Confocal and electron microscopy studies using M. bovis mNeonGreen strain were also performed. All 14 Mycoplasma strains multiplied within the MDBK cells, a finding confirmed by microscopy studies of the M. bovis mNeonGreen strain. Unexpectedly, the M. bovis strains, obtained from diseased and asymptomatic cattle and bison, had lower Myc/Cell ratios than M. bovirhinis and M. bovigenitalium strains. These findings suggest that the ability for mycoplasmas to invade and replicate within host cells does not account for the differences in virulence between species.
Insights
This study compared the cellular invasion of cattle Mycoplasma species. Unexpectedly, Mycoplasma bovis strains showed lower invasion and replication in kidney cells than other species, challenging virulence assumptions.
Area of Science:
- Veterinary Microbiology
- Bovine Pathology
- Cellular Biology
Background:
- Mycoplasma bovis is a significant cattle pathogen.
- Other Mycoplasma species like M. bovigenitalium and M. bovirhinis are also linked to bovine mycoplasmosis.
- Understanding species-specific virulence factors is crucial for disease control.
Purpose of the Study:
- To compare the adhesion and cellular invasion of different bovine Mycoplasma species.
- To investigate the intracellular replication of Mycoplasma bovis, M. bovigenitalium, and M. bovirhinis in Madin-Darby Bovine Kidney (MDBK) cells.
- To correlate in vitro cellular interactions with observed virulence.
Main Methods:
- Infection of MDBK cells with 12 M. bovis strains and one strain each of M. bovigenitalium and M. bovirhinis.
- Gentamicin protection assay to quantify intracellular Mycoplasma at 6 and 54 hours post-infection.
- Quantitative PCR (qPCR) to determine Mycoplasma/cell ratios and microscopy for visualization.
Main Results:
- All tested Mycoplasma strains, including M. bovis, multiplied within MDBK cells.
- Unexpectedly, M. bovis strains exhibited lower intracellular Mycoplasma/cell ratios compared to M. bovigenitalium and M. bovirhinis.
- Microscopy confirmed intracellular replication of M. bovis.
Conclusions:
- Intracellular invasion and replication capabilities within MDBK cells do not fully explain the observed virulence differences among bovine Mycoplasma species.
- Further research is needed to identify other virulence factors contributing to Mycoplasma-associated diseases in cattle.
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